Three novel types of splicing aberrations in the tuberous sclerosis TSC2 gene caused by mutations apart from splice consensus sequences

Disease causing aberrations in both tuberous sclerosis predisposing genes, TSC1 and TSC2, comprise nearly every type of alteration with a predominance of small truncating mutations distributed over both genes. We performed an RNA based screening of the entire coding regions of both TSC genes applyin g the protein truncation test (PTT) and identified a high proportion of unu sual splicing abnormalities affecting the TSC2 gene. Two cases exhibited di fferent splice acceptor mutations in intron 9 (IVS9-15G-->A and IVS9-3C-->G ) both accompanied by exon 10 skipping and simultaneous usage of a cryptic splice acceptor in exon 10. Another splice acceptor mutation (IVS38-18A-->C ) destroyed the putative polypyrimidine structure in intron 38 and resulted in simultaneous intron retention and usage of a downstream cryptic splice acceptor in exon 39. Another patient bore a C-->T transition in intron 8 (I VS8+281C-->T) activating a splice donor site and resulting in the inclusion of a newly recognised exon in the mRNA followed by a premature stop. These splice variants deduced from experimental results are additionally support ed by RNA secondary structure analysis based on free energy minimisation. T hree of the reported splicing anomalies are due to sequence changes remote from exon/intron boundaries, described for the first time in TSC. These fin dings highlight the significance of investigating intronic changes and thei r consequences on the mRNA level as disease causing mutations in TSC. (C) 2 000 Elsevier Science B.V. All rights reserved.