Abstracts

Rationale: Somatic genetic variants are the most common cause of intractable pediatric focal epilepsies and frequently associated with malformations of cortical development such as focal cortical dysplasia (FCD). Histopathological hallmarks of FCDs are cortical mislamination and the presence of morphologically abnormal cells such as dysmorphic neurons (DNs) and balloon cells (BCs) which are indicative of pathogenic variants activating the PI3K-mTOR signaling pathway. Electrophysiologic studies have shown that DNs are inherently hyperexcitable, but the identities of DNs and BCs is still debated and the mechanisms through which a small variant-positive cell population causes disease is unknown.


Methods: To examine the transcriptional changes in FCD we performed single nucleus RNA sequencing (snRNA-seq) on 20 surgically resected FCD samples, representing a range of pathogenic variants in PIK3CA, MTOR, DEPDC5, etc., and 8 neurotypical postmortem control samples. Given that only a minority of cells in surgically resected FCD samples harbor the pathogenic variants, we wished to evaluate gene expression changes in variant-positive and variant-negative cells separately. In order to elucidate the cell autonomous mechanisms of somatic variants in FCD, we developed GoTEN, which combines 10X snRNA-seq with Oxford Nanopore (ONT) long-read amplicon sequencing. We applied GoTEN to select FCD samples to simultaneously obtain the genotype and the transcriptional states associated with variant-positive and variant-negative cells.


Results: Differential gene expression analysis in cases vs controls showed significant and likely compensatory downregulation of gene pathways downstream of mTORC1 signaling in all the major cell types, but most significantly in neurons. Moreover, the FCD-associated microglia exhibited a transcriptional program consistent with a pro-inflammatory state with corresponding activation of immune-related gene pathways in astrocytes and oligodendrocytes. Surprisingly, no new cell clusters representing DNs or BCs were identified and all major cell type proportions were unaffected in the FCD tissue. We applied GoTEN to 3 samples harboring PIK3CA and MTOR variants. GoTEN analysis detected the pathogenic variants mostly in excitatory and in some inhibitory neurons, although a small percentage of variant-positive glial cells were also seen, which is consistent with shared neuroglial progenitors. Strikingly, the variant-positive cells had indistinguishable cellular identities relative to their variant-negative counterparts although they did have disease-associated transcriptional changes mostly restricted to PI3K-mTOR pathway genes.

Conclusions: In summary, using a combination of snRNA-seq and a new method, GoTEN, our study highlights genotype-specific transcriptional changes and a pro-inflammatory state in FCD which could be potential targets for precision therapies in FCD. Additionally, despite drastic morphologic changes in situ, we find little to no change in variant-positive cell identities.


Funding: 1. NINDS K08-NS128272
2. NINDS R01-NS035129
3. Doris Duke Physician Scientist Fellowship
4. Burroughs Wellcome Fund Career Award for Medical Scientists
5. Howard Hughes Medical Institute