Abstracts

Rationale: Structural variants (SVs) are genetic variants that range from 50 bp to a few megabases in size and include deletions, duplications, insertions, inversions and translocations. SVs account for up to 12% of genetic epilepsies. Some SVs overlap known epilepsy-associated genes and many of these cause epilepsy through gene dosage effects. Some SVs do not span coding regions of the genome, but rather can disrupt cis-regulatory regions, or higher order chromatin structures, including topologically associated domains (TADs). These loci are important for 3D genome architecture and gene expression regulation. When native TADs are disrupted, the chromatin is rearranged, and genes encompassed by the original TAD may be influenced by effectors within the newly formed TAD (neo-TAD). Such rearrangements can cause unfavorable gene misregulation. We hypothesize that some epilepsy-associated SVs disrupt TADs leading to altered gene expression of genes important in epilepsy.

Methods: We identified a microdeletion in the 14q12 region overlapping the TAD boundary that contains FOXG1 in individuals with epilepsy and associated neurodevelopmental disorders. Forkhead Box G1 (FOXG1) is an important transcription factor involved in mammalian cerebral corticogenesis, and FOXG1 haploinsufficiency is associated with epilepsy. We hypothesize that the 14q12 microdeletion disrupts TAD boundaries at the FOXG1 locus, leading to neoTAD formation and FOXG1 misexpression. We will utilize iPSC derived neuronal models for our studies as FOXG1 expression is highest in neuronal cell types. We will introduce this microdeletion via CRISPR based genome editing approaches to create a stable iPSC cell line with an appropriate isogenic control line, and reprogram the iPSCs to neurons to study the molecular effects of the proposed variants. We will investigate TAD architecture using HiC in the presence and absence of the microdeletion in iPSC derived neurons, and evaluate FOXG1 expression using RT-PCR and RNA-Seq. We will also investigate cellular phenotypes resulting from FOXG1 misregulation in these neurons.

Results: Fibroblasts from a 25-year-old healthy individual (GM03651, Coriell Institute) have been reprogrammed to generate induced pluripotent stem cells (iPSCs). Using CRISPR-Cas9 editing, we have successfully introduced the 14q12 microdeletion in iPSCs. We designed qRT-PCR probes and detected FOXG1 expression in iPSCs. We have also identified TAD boundaries across the genome, and Hi-C probes targeting these boundary regions are currently being designed.

Conclusions: This study will be the first of its kind to explore molecular mechanisms of non-coding SVs associated with epilepsy, by exploring changes in chromatin architecture to investigate genetic etiology of epilepsy. The strategy proposed here can be extended to explain the effect of SVs around other epilepsy related genes, and can be extrapolated to help explain the effects of SVs in other genetic diseases as well.

Funding: Please list any funding that was received in support of this abstract.: This research is supported by the Chicago Biomedical Consortium Award presented to Dr. Gemma L. Carvill and Dr. Marcelo A. Nobrega.