Illegitimate splicing of the NF1 gene in healthy individuals mimics mutation-induced splicing alterations in NF1 patients

Neurofibromatosis type 1 (NF1) is a common inherited disease affecting one in 3500 individuals. The mutation rate in the NF1 gene is one of the highes t known for human genes. Compared to other methods, the protein truncation test (PTT) and subsequent sequence analysis of cloned cDNA provides improve d efficiency in detecting NF1 mutations that are dispersed throughout the g ene spanning 350 kb of genomic DNA. Sequencing of cDNA of patients affected with NF1 mutations revealed multiple splicing errors. Since similar misspl icings were also found in "aged" blood of healthy individuals, they are mos t likely attributable to a general decrease in splice site selection in age d blood. We show that restoring viability of lymphocytes before RNA extract ion by cultivation and PHA stimulation diminishes aberrant splicing in aged blood and is thus useful to circumvent splicing alterations which are freq uently compromising mutation detection in patient samples and mimic mutatio n-induced alterations of mRNA.