Abstract
Clinical sequencing of a vaginal adenocarcinoma specimen yielded a reported TSC2 variant
which was classified as splice-modulating loss-of-function, prompting consideration of a clinical
trial for tumors with TSC2 loss. Literature review suggested that germline alterations affecting
this particular exon are considered non-pathogenic, raising concern about the initial somatic
variant interpretation. To resolve this ambiguity, raw data were provided by Tempus and an
integrated DNA & RNA analysis allowed detailed characterization of the impact of this alteration
on splicing. We confirm that this variant results in the complete exclusion of exon 26 from the
transcript, however the exon 25-27 splice results in an in-frame transcript. Analysis of many
additional wild-type tumor and normal tissue samples demonstrate that exon 26 is commonly
spliced out of TSC2 transcripts, indicating that the transcript containing the exon 25-27 splice is
highly unlikely to be pathogenic. This analysis did not support the classification of this variant as
pathogenic and suggests that patients with such mutations should not be candidates for
therapeutics designed for TSC2 dysfunction.

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