Patient TSC2 Mutant Cells Exhibit Aberrations in Early Neurodevelopment Accompanied by Changes in the DNA Methylome

Tuberous Sclerosis Complex (TSC) is a debilitating developmental disorder characterized by a variety of clinical manifestations. While benign tumors in the heart, lungs, kidney, and brain are all hallmarks of the disease, often the most severe symptoms of TSC are neurological, including seizures, autism, psychiatric disorders, and intellectual disabilities. TSC is caused by a heterozygous loss of function mutation in TSC1 or TSC2 and consequent dysregulation of signaling via mechanistic Target of Rapamycin Complex 1 (mTORC1). While TSC neurological phenotypes are well-documented, it is not yet known how early in neural development TSC1/2-mutant cells diverge from the typical developmental trajectory, and whether such phenotypes are seen in the heterozygous-mutant populations comprising the vast majority of cells in patient tissues. Using TSC patient-derived isogenic induced pluripotent stem cells (iPSCs), we observed aberrant early neurodevelopment in vitro, including misexpression of key transcription factors associated with lineage commitment and premature electrical activity. These alterations in differentiation were coincident with widespread changes in DNA methylation, including at loci associated with key genes in neurodevelopment. Collectively, these data suggest that mutation or loss of TSC2 affects gene regulation and expression at earlier timepoints than previously appreciated, with implications for whether and how prenatal treatment should be pursued. Overall design: To investigate gene expression differences between patient heterozygous TSC2 mutant day 10 NPCs and isogenic wild type controls or homozygous TSC2 mutants, we extracted total RNA, sequenced, and analysed differences between genotypes. Two separate isogenic iPSC lines were used in this study. TSP77 is patient derived while CR39/37 are not. Two separate differentiations were used per cell line as replicates.