90% TSC1/TSC2 mosaicism detection rate in Tuberous Sclerosis Complex patients without mutation identified in commercial labs

Principal Investigator: David J Kwiatkowski

Authors: Katarzyna Klonowska, Joannes Grevelink, Krinio Giannikou, Magdalena Tyburczy, David J Kwiatkowski
Lay Abstract

Tuberous sclerosis complex (TSC) is a human genetic disorder due to mutations in the TSC1 or TSC2 genes, characterized by distinctive tumors in multiple body sites. Genetic analysis [Massively Parallel Sequencing (MPS)] performed in our laboratory revealed that mosaicism is common in TSC patients who had ‘no mutation identified’ (NMI) in commercial clinical labs. Mosaicism is a situation in which different cells in the body have a different genetic make-up; in TSC, it occurs due to a TSC1/TSC2 mutation event resulting in a population of cells in the body with the TSC1/TSC2 variant, mixed in with a larger fraction of cells without the variant.

In this study, we have developed MPS-based strategy for very sensitive TSC1/TSC2 mutation detection that is capable of a 90% detection rate on NMI cases. We used our MPS strategy for the analysis of 144 samples derived from different TSC tumors/tissues/fluids, from 30 NMI TSC patients. The analysis confirmed the robustness of our approach for TSC1/TSC2 mutation detection. It enabled detailed characterization of the spectrum of mosaic mutations in TSC, which may occur at different level [variant allele frequency (VAF)] in different body sites (blood VAF:0-19%, median:2.8%) and are enriched in TSC-related tumors.

Scientific Abstract

Introduction: Our past Massively Parallel Sequencing (MPS) revealed that mosaicism is common in Tuberous Sclerosis Complex (TSC) patients who had ‘no mutation identified’ (NMI) in commercial labs.

Materials and Methods: We performed analysis of 144 samples [different TSC tumors/tissues/fluids] from 30 NMI TSC patients, using MPS and our new Multiplex High-sensitivity PCR Assay (MHPA) enabling MPS error suppression [sensitivity:0.05% variant allele frequency (VAF)].

Results: TSC1/TSC2 mutations were identified in 27 of 30 patients (90%) [21(78%) in TSC2; 6(22%) in TSC1]; 25 patients had mosaicism [blood VAF:0-19%, median:2.8%]. VAFs of the mosaic mutations were enriched in TSC tumors in comparison with normal tissues/fluids. We identified 6 novel TSC1/TSC2 mutations, including 4 large mutations, a large (221kb) inversion, and a de novo deep intronic deletion. We also identified an extraordinary case of an individual with minimal TSC features and two unique TSC2 mutations in TSC-related angiomyolipoma and angiofibroma tumors, suggesting that both tumor events were sporadic.

Conclusions: We have developed a strategy for very sensitive TSC1/TSC2 mutation detection that is capable of a 90% detection rate on NMI cases and defined the spectrum of mosaic mutations in TSC.

Funding: Engles Family Fund (DJK) and FY2020 TSC Alliance Postdoctoral Fellowship Award (KK)

Clinical Implications
We developed a novel strategy for very sensitive detection of variants in the TSC1/TSC2 genes related to Tuberous Sclerosis Complex (TSC); it is capable of a 90% detection rate on TSC patients who had no variant identified in clinical labs.

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