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Three novel types of splicing aberrations in the tuberous sclerosis TSC2 gene caused by mutations apart from splice
K Mayer1, W Ballhausen, W Leistner
1Institute of Human Genetics, University of Erlangen-Nuremberg, Erlangen, Germany. kmayer@humgenet.uni-erlangen.de
Abstract:
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 applying the protein truncation test (PTT) and identified a high proportion of unusual splicing abnormalities affecting the TSC2 gene. Two cases exhibited different 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-->G) 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 (IVS8+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 supported by RNA secondary structure analysis based on free energy minimisation. Three of the reported splicing anomalies are due to sequence changes remote from exon/intron boundaries, described for the first time in TSC. These findings highlight the significance of investigating intronic changes and their consequences on the mRNA level as disease causing mutations in TSC.
Insights
Tuberous sclerosis complex (TSC) is often caused by mutations in TSC1 and TSC2 genes. This study found unusual splicing abnormalities in the TSC2 gene, highlighting the importance of examining intronic changes for disease-causing mutations.
Area of Science:
- Genetics
- Molecular Biology
- Biochemistry
Background:
- Tuberous sclerosis complex (TSC) is a genetic disorder caused by mutations in the TSC1 and TSC2 genes.
- Mutations in these genes can lead to various alterations, with a prevalence of small truncating mutations.
Purpose of the Study:
- To investigate RNA-based alterations in the TSC1 and TSC2 genes.
- To identify novel splicing abnormalities contributing to TSC pathogenesis.
Main Methods:
- RNA-based screening of TSC1 and TSC2 coding regions using the protein truncation test (PTT).
- Analysis of splice acceptor and donor site mutations, exon skipping, intron retention, and cryptic splice site usage.
- RNA secondary structure analysis to support experimental findings.
Main Results:
- A high proportion of unusual splicing abnormalities were identified in the TSC2 gene.
- Specific mutations were found to cause exon skipping, intron retention, and cryptic splice site activation.
- Novel splicing anomalies, including those distant from exon-intron boundaries, were discovered.
Conclusions:
- Splicing abnormalities represent a significant class of disease-causing mutations in TSC, particularly within the TSC2 gene.
- Intronic sequence changes and their impact on mRNA splicing are crucial for understanding TSC.
- Further investigation into intronic mutations is warranted for comprehensive TSC genetic analysis.