Tuberous sclerosis type 2: RNA based detection of complex splicing aberrations in the TSC2 gene by the protein truncation test (PTT). K. Mayer, S. Schirdewahn, W. Ballhausen, HD. Rott. Institute of Human Genetics, Erlangen, Germany.

   Mutation analyses in tuberous sclerosis (TSC) have reported a wide variety of disease causing aberrations in the two known predisposing genes, TSC1 and TSC2 on chromosomes 9q34 and 16p13, comprising mainly small truncating mutations distributed over the entire genes. We performed an RNA based screening of the entire coding regions of both TSC genes applying the protein truncation test (PTT) and identified a high proportion of unusual splicing abnormalities affecting the TSC2 gene. Aberrant splicing products were initially detected as shortened polypeptides after in vitro transcription/translation, were identified through cloning and sequencing, and were confirmed by direct amplification of RT-PCR products. In two representative examples the causative changes on the genomic level turned out to be different splice acceptor mutations in intron 9 (IVS9-15G>A and IVS9-3C>G) both leading to identical exon 10 skipping and simultaneous usage of a cryptic splice acceptor in exon 10. Another splice acceptor mutation (IVS38-18A>G) destroyed a branch point sequence in intron 38 and resulted in simultaneous intron 39 retention and usage of a downstream cryptic splice acceptor in exon 39. All splice acceptor mutations described lead to different types of abnormal truncated transcripts deleting or affecting one of the known interaction domains of tuberin either for hamartin or for rabaptin5. One further patient exhibited a C>T transition in intron 8 (IVS8+281C>T) activating a consensus splice site and resulting in inclusion of a newly recognized exon in the mRNA followed by a premature stop. It is important to note that three of the reported splicing abnormalities are due to sequence changes remote from exon/intron boundaries which are described for the first time in TSC. These findings strengthen the importance of investigating intronic changes and their consequences on the mRNA level as disease causing mutations in TSC.