Identifying mosaic genetic variants in tuberous sclerosis complex

Abstract

Tuberous sclerosis complex (TSC) is an autosomal dominant condition caused by pathogenic variants in the TSC1 or TSC2 gene with a prevalence of 8.8 per 100 000. TSC is associated with variable neurodevelopmental outcomes, seizures and benign tumours in various organs. Around 10-15% have no pathogenic variants identified – the “no mutations identified” (NMI) cohort. Previously, this cohort were reported to be phenotypically milder and have mosaic/intronic variants. Many have reproductive concerns, but options are not open to them without a molecular diagnosis. This study aims to determine the phenotype of an Australian NMI cohort, and if testing multiple samples of an individual would allow for an improved diagnostic yield using a testing strategy that may be viable in a diagnostic laboratory. The first study discussed the phenotype of the NMI cohort in the Sydney Children’s Hospital TSC management clinic, a tertiary referral centre. A medical records review was used to compare the clinical picture of those in the NMI, heterozygous, and mosaic cohorts. This showed that although those in the NMI cohort likely have a milder neurodevelopmental outcome, they are no different to the heterozygous cohort when considering the number of organ systems involved. This differs from the previous literature. The second study explored the use of multiple samples in deep sequencing of the NMI cohort. Massively parallel sequencing (MPS) using a custom target capture panel consisting of the whole genomic region of TSC1 and TSC2 was performed. A minimum of 2 samples was tested for each participant, including affected tissue where possible. Sequencing occurred with a target read depth of 500x initially and proceeded to 4000x for those who remained persistently NMI. A diagnostic yield of 72% was achieved in the probands tested, with the majority being mosaic variants and the remainder missed heterozygous variants. The use of multiple samples allowed for validation of otherwise discarded low-level mosaic variants. In conclusion, this thesis explored the phenotype and genotype of the NMI cohort. It showed that those in this cohort may not be as mild as previously suggested. Testing multiple samples allows for detection of germline mosaic variants on MPS without excessive cost and the need for specialised techniques. Scaling this up to diagnostic testing is likely viable, which would ultimately be critical for optimal clinical care of the NMI cohort