Abstracts

Rationale: Malformations of cortical development (MCD) arise from defects in neuronal migration, cellular proliferation, or cortical organization. These heterogeneous disorders are characterized by abnormalities in the structure and organization of the cerebral cortex, leading to intractable epilepsy and intellectual disability. While approximately 30% of cases of MCD can be attributed to a de novo somatic mutation in the PI3K-AKT-mTOR pathway, the vast majority of cases have no known cause. To that end, we sought to identify novel somatic mutations in hemimegalencephaly (HME), focal cortical dysplasia (FCD), and polymicrogyria (PMG), which are three subtypes of MCD commonly encountered in patients with pediatric epilepsy, taking into account the patients’ clinical, pathological, and radiological phenotypes Methods: To identify somatic mutations, we performed paired exome sequencing and/or targeted sequencing of surgically resected brain tissue and leukocytes from 30 individuals with HME, FCD or PMG. Sequencing libraries were prepared with either the Illumina TruSeq or Kapa library preparation kit; targeted capture of the exome was performed with either Illumina TruSeq or Roche SeqCap EZ Exome v3.0 exome enrichment kits, while targeted capture of the 192 genes in the PI3K-AKT-mTOR pathway was carried out with a custom Roche SeqCap EZ kit. Paired-end sequencing reads were generated on either an Illumina HiSeq 2000 or HiSeq 2500. Somatic single nucleotide variants and indels were called with Mutect2 and Strelka; somatic loss of heterozygosity was assessed with Varscan-2. Likely pathogenic variants, i.e., those that were predicted to have a deleterious effect on the final protein and that were absent from in-house controls and public databases, were confirmed with digital droplet PCR or amplicon sequencing. Results: The sequencing coverage was greater than 200-fold and 800-fold, on average, for the samples sequenced with exome sequencing and targeted sequencing, respectively. We have identified and confirmed five somatic point mutations in known genes. Two were confirmed in MTOR (NM_004958.3:c.133C>A;p.Gln45Lys and NM_004958.3:c.4447T>C;p.Cys1483Arg]) in individuals with FCD and HME, respectively. Two were confirmed in PIK3CA (NM_006218.2:c.1624G>A;p.Glu542Lys and NM_006218.2:c.1633G>A;p.Glu545Lys) in individuals with HME with PMG (Figure 1A) and FCD, respectively. The p.GLu542Lys mutation has previously been reported to be selectively present in abnormal non-brain tissue patients with CLOVES (Kurek KC et al. Am J Hum Genet. 2012), demonstrating that differences in the timing and location of somatic mutations give rise to variable phenotypes. Additionally, one mutation was confirmed in TSC1 (NM_000368.4:c.298C>T;p.Gln100*) in a patient with FCD (Figure 1B). Additional candidates in novel genes are in the process of being confirmed. Conclusions: The confirmed mutations identified thus far further support the role for altered regulation of the mTOR pathway as a pivotal pathogenic mechanism for many MCD subtypes and suggest overlapping pathophysiology among these subtypes. Funding: This study was supported by NINDS R01NS094596 (E.L.H.and P.B.C.), and R01NS082343-01 (P.B.C.) and Citizens United for Research in Epilepsy (CURE) (P.B.C.)