Biochemical characterisation of TSC1 and TSC2 variants identified in patients with tuberous sclerosis complex

After identification of TSC1 and TSC2, the genes that are mutated in
individuals with tuberous sclerosis (TSC), we demonstrated that the TSC1 and
TSC2 gene products form a protein complex. We characterised this complex and
demonstrated that pathogenic missense mutations disrupt the complex. In
addition, we performed mutation analysis on a cohort of >650 TSC index
patients. In the majority of cases we identified the pathogenic mutation.
However, in >50 cases it was impossible to ascertain from the genetic data
alone whether specific sequence changes were pathogenic. We labelled these
changes 'unclassified variants'.
Since the identification of the role of the TSC1-TSC2 complex in regulating
signal transduction through mTOR, and the demonstration that the complex is a
GTPase activating protein (GAP) for rheb, we have analysed the effect of
different unclassified TSC1 and TSC2 variants on the activity of the TSC1-TSC2
complex. This has the potential to extend the diagnostic service that can be
offered to TSC patients and their families.

Pathogenic, non-truncating TSC1 and TSC2 mutations can be distinguished from
non-pathogenic changes by studying their effects on the TSC1-TSC2 protein
complex, and on splicing of the TSC1 and TSC2 mRNAs. Functional
characterisation of unclassified TSC1 and TSC2 variants will complement
existing diagnostic tests and enable appropriate clinical care and counselling
for more TSC patients and their relatives. We hypothesise that specific
non-truncating TSC1 and TSC2 mutations will be associated with a mild TSC
phenotype. Characterisation of multiple TSC1 and TSC2 variants will also help
define the structural and catalytic domains in TSC1 and TSC2.

Observational study.

Each participant has provided a blood sample for the isolation of genomic DNA
as part of the standard genetic consultation procedure. TSC1 and TSC2 mutation
screening is performed by the Section of DNA Diagnostics within the Department
of Clinical Genetics at the Erasmus Medical Center. The mutation screening
procedure is not part of the research project. In approximately 20 specific
cases where a potential splice site mutation, or a mutation affecting
transcription, is identified, a skin biopsy (3 mm) will be requested. This is a
very minor surgical procedure carried out under local anaesthetic with
negligable rsks to the patient. Temporary discomfort and some scar tissue are
possible.
The study is non-therapeutic. However, a potential benefit is that patients and
their relatives will obtain better information regarding their TSC1 or TSC2
mutation status.