Neurofibromatosis type 1 (NF1) is a common autosomal dominant syndrome with complete penetrance, but a broad spectrum of clinical expression. It is caused by mutation in the NF1 gene. Mutation analysis of this 60-exon gene is complicated due to its size and complexity, the existence of a number of highly similar pseudogenes and a broad spectrum of different mutations in patients with the occurrence of many unusual splicing mutations. RNA-based mutation analysis that is based on sequence analysis of long-range reverse transcription polymerase chain reaction (RT-PCR) products covering the entire coding region (direct cDNA-sequencing) largely overcomes these difficulties and, hence, allows NF1 mutation analysis with highest sensitivity and specificity. Here we render a detailed description of the technique starting from short-term phytohemagglutinin (PHA)-stimulated and puromycin-treated lymphocyte cultures to RT-PCR amplification and subsequent sequence analysis of the resulting fragments. We give advice on how to interpret splicing alterations found by this method and how to detect different types of NF1 splicing mutations. © 2012 by Nova Science Publishers, Inc. All rights reserved.
