The NF1 gene is a large and complex gene spread over 280 kb of genomic DNA on chromosome 17q11.2 and comprising 57 constitutive and at least 3 alternatively spliced exons. The NF1 gene encodes a transcript with an open reading frame of 8,454 nucleotides. Mutation detection is a challenge due to the large size of the gene, the lack of mutational hotspots, the occurrence of a very diverse spectrum of mutation types and the presence of more than 30 unprocessed pseudogenes . Several different techniques have been applied to look for mutations in this complex gene [2-4], which included already early on attempts to apply RNA-based assays, i.e. protein truncation testing (PTT) . We initially started out using this PTT assay [6, 7], but soon realized its limitations which are (i) the necessity for complementary assays capable to detect non-truncating mutations and (ii) the need to overcome the problems inherent to all RNA-based approaches, i.e. illegitimate splicing and nonsense mediated RNA decay. However, we realized the enormous advantages of an RNA-based mutation analysis approach for a large and complex gene like NF1 that is expressed in the lymphocytes, that has no hotspots for mutations, where mutations of all types exist and where multiple highly homologous unprocessed pseudogene sequences that are spread over the genome hamper specific DNA amplification. Therefore, we further refined and improved the assays so that they now represent the core of a comprehensive mutation analysis cascade that allows highly sensitive and effective NF1 testing. In the following, we will present this approach and the mutational spectrum deduced from its application to a large cohort of patients. Copyright © 2008 S. Karger AG, Basel.