Abstracts

Rationale: Hemimegalencephaly (HMEG) is a rare pediatric malformation of cortical development caused by somatic mutations in the PI3K-AKT-mTOR signaling pathway that often leads to drug-resistant epilepsy and intellectual disability. While the activation of mTOR is well-documented in these patients, the variable clinical response to the mTOR inhibitor rapamycin highlights gaps in our understanding of the disease mechanism.


Methods: This study employs SoMoSeq (Somatic Mosaicism Sequencing), a novel technique that combines genotype-informed single-cell genotyping and RNA sequencing, to analyze surgically resected brain tissue from HMEG patients with a known pathogenic somatic PIK3CA mutation (E545K). The goal of this study is to identify specific cell types carrying the mutation and to characterize their associated transcriptional signatures.


Results: We performed SoMoSeq on 4,300 nuclei from brain tissue of three HMEG patients with variant allele frequencies (VAF) ranging from 25-14%, comparing them to age, sex, and region-matched neurotypical autopsy controls. Single-cell genotyping identified three distinct cellular populations: heterozygous (~44-28% cells), homozygous variant (~6-2% cells), and homozygous wild type (~50-70% cells) which was confirmed through whole genome amplification followed by Sanger sequencing in two of the three cases (third case pending analysis). Initial findings indicate the presence of loss of heterozygosity (LOH) on chromosome 3 in a subset of cells. This finding is being further examined using primary template amplification and genome-wide SNP arrays to precisely map LOH coordinates. Additionally, we are analyzing single-cell RNA sequencing data to investigate the distribution and transcriptional profiles of these populations across common brain cell types. A mixed-effects model is being used to evaluate the influence of genotype, case-control status, and cell type on gene expression.


Conclusions: The presence of LOH in a subset of cells aligns with Knudson's two-hit hypothesis, indicating that secondary genetic events, such as LOH, could play a significant role in the pathogenesis of HMEG alongside the initial somatic mutation. These insights into the cellular mechanisms of HMEG, particularly the cell-autonomous and non-cell-autonomous effects of PIK3CA variants, provide a deeper understanding of the disease and may inform the development of more effective targeted therapies.


Funding: Funding: This study was supported by R01NS094596, R21NS133873