Abstracts

Rationale:
Megalencephaly (MEG), Hemimegalencephaly (HMEG) and Focal Cortical Dysplasia (FCD) are neurodevelopmental disorders characterized by brain overgrowth and cortical abnormalities, associated with significant pediatric morbidity and mortality including epilepsy, autism and intellectual disability. Importantly, FCD is the most common cause of intractable pediatric focal epilepsy. Gain and loss of function mutations in the PI3K-AKT-MTOR pathway have been identified in this spectrum, with variable levels of mosaicism and distribution of causal mutations; however, mechanisms underlying brain overgrowth, dysplasia, and epileptogenesis are not well understood. We aimed to better define the genotype-phenotype correlation among the most common hotspot mutations in the pathway.
Method:
We collected multiple samples in a cohort of 128 patients (370 samples, 299 epilepsy brain tissue) and tested them using digital droplet PCR (ddPCR). We subsequently modeled disease pathogenesis in vitro using patients-derived cells: we generated induced pluripotent stem cells carrying the common PIK3CAH1047R and MTORT1977I mutations and differentiated them to Neuronal Progenitor Cells (NPCs) and cerebral organoids. We performed functional assays including population doubling time, analysis of cellular morphology and size, differentiation and cortical layering.
Results:
Using ddPCR, we were able to solve 14% of our cohort (18 patients), with a diagnostic yield of 37% in HMEG and 12.7% in FCD. Interestingly, individuals with FCD mostly had mutations in MTOR, while those with MEG and HMEG had PIK3CA mutations. Further, among FCD cases, only FCD type 2 were associated with mutations in the pathway. For the first time our data provide the link between AKT3 mutations to non-FCD epilepsy, namely mesial temporal sclerosis, in two unrelated patients. Our preliminary results show overlapping and exclusive cellular phenotypes in PIK3CA and MTOR mutant cell lines. Specifically, both mutant lines had increased NPCs proliferation and hypertrophy, albeit PIK3CA mutants displayed the most severe phenotype. MTOR and PIK3CA mutant organoids had an average area at least double that of controls, recapitulating MEG in vitro, with abnormal organoid morphology suggesting neuronal migration defects. In addition, MTOR mutant lines displayed Outer Radial Glia cells hyperplasia and dysplasia, suggesting a novel mechanism underlying MTOR-related megalencephaly.
Conclusion:
In summary, our results show that distinct mechanisms underlie PIK3CA- and MTOR- related MEG and FCD during human embryonic development. As a future direction, we will expand our studies to include AKT3 mutant cell lines and to test select MTOR pathway inhibitors (e.g. Rapamycin and Alpelisib). This work will help characterize mechanisms of MEG and FCD caused by mutations in MTOR pathway and guide the design of future clinical trials.
Funding:
:NINDS K08 NS092898 (GMM) UC Davis (FP,GMM) NINDS R01 NS092772-05 (WBD) BBI Catalytic Collaborations Award (GMM, JH) BBI Precision Medicine Clinical Labs Award (GMM)